Methods and compositions for regulating the intravascular flow and oxygenating activity of hemoglobin in a human or animal subject

ABSTRACT

Methods for time-regulated prophylaxis or treatment of animals or humans for limited circulatory oxygen delivery induced by the inhibitory effects of a plasma-borne hemoglobin-based material on L-arginine→nitric oxide→cGMP pathways in the arteriovenous vasculature. The properties of the invention restore and increase circulatory oxygen delivery by increasing circulatory flow of the blood-hemoglobin-based material through selective activation of L-arginine→nitric oxide→cGMP pathways in the arterial rather than venous vasculature. A method of the invention utilizes oxygen-carrying biocolloid compositions that consist of a hemoglobin-based material and a guanosine 3′:5′-cyclic monophosphate (cGMP) generating entity, for treatment of animals and humans in need thereof for diseases or medical conditions which utilize the biocolloids as hemodiluents, blood substitutes, plasma expanders, or resuscitative fluids. The invention provides selective administration of cGMP generating entities for prophylaxis or treatment of animals or humans with limited circulatory oxygen delivery induced by a plasma hemoglobin-based material arising from intravenous administration, disease or medical condition. Most importantly, the invention provides for time-controlled enablement of the oxygen-deliverying properties of the invention that would be used for treatment of specific diseases or medical conditions requiring time-dependent increases in circulatory oxygen delivery.

This is a continuation of patent application Ser. No. 07/849,610, filedMar. 11, 1992 and now abandoned, the disclosure of which is herebyincorporated herein in its entirety.

A portion of the disclosure of this patent document contains materialwhich is subject to copyright protection. The copyright owner has noobjection to the facsimile reproduction by any one of the patentdisclosure, as it appears in the Patent & Trademark Office patent filesor records, but otherwise reserves all copyright rights whatsoever.

FIELD OF THE INVENTION

The present invention relates generally to compositions that enhance thein vivo oxygenating properties of hemoglobin products. Moreparticularly, the present invention relates to time-controlledsuperoxygenating compositions that comprise hemoglobin colloid andguanosine 3′:5′-cyclic monophosphate (cyclic GMP) generating compounds,and to methods for treatment of diseases or medical conditions whichutilize the time-controlled superoxygenating compositions asbiocolloids, i.e. hemodiluents, blood substitutes, plasma expanders, orresuscitative fluids.

BACKGROUND

There are many medical conditions, for example hemorrhagic hypotensionand anaphylactic shock, in which significant blood loss and/orhypotension (abnormally low blood pressure) occur leading to reducedtissue oxygenation. For patients with such medical conditions, it isdesirable and often critical for their survival to stabilize their bloodpressure and to increase the amount of oxygen provided to body tissuesby their circulatory systems.

Considerable effort has therefore been expended in developing colloidalsubstances which may be used as resuscitation fluids and/or blood plasmaexpanders for stabilizing blood pressure by hemodilution (i.e.,increasing blood plasma volume) and which are capable of carrying anddelivering oxygen to bodily tissues. The costs, risks (includingcontamination with disease-causing viruses) and histocompatibilityrequirements associated with the transfusion of whole blood or bloodfractions have stimulated researchers to develop alternateoxygen-carrying substances.

Hemoglobin, the natural respiratory protein of erythrocyte which carriesoxygen to body tissues from the lungs, is a potential alternateoxygen-carrying biocolloid. Erythrocytes contain approximately 34 gramsof hemoglobin per 100 ml of red cells.

Hemodilution experiments with hemoglobin have revealed that unlikehemodilution with albumin, hemodilution with hemoglobin does not augmentcardiac output (whole-body blood flow). During hemodilution with inertcolloids such as albumin, whole-body blood flow increases inverselyproportional to the level of hemodilution. In other words, an ≈50%hemodilution (i.e., an ≈50% decrease in blood red cell mass) increasesblood flow ≈100%, and so on. The physical basis of the hematocrit-bloodflow inverse relationship is analogous to viscosity-flow mechanics. Inother words, decreases in hematocrit cause decreases in blood viscositywhich results in increases in blood flow. Hemoglobin, on the other hand,is not inert. Hemodilution with hemoglobin causes vasoconstriction whichresults in smaller diameter blood vessels. Therefore, even though bloodviscosity is decreased during hemodilution with hemoglobin, the smallerdiameter blood vessels resist increases in blood flow. This is analogousto direct mechanical relationship between flow and tube diameter, i.e.,decreased diameter results in decreased flow. Whole-body flow (cardiacoutput) is not increased during hemodilution with hemoglobin becauseflow of the less viscous blood is opposed by the hemoglobin-mediateddecrease in blood vessel diameter.

Increased cardiac output is desirable to increase oxygen delivery to thetissues. Examples of publications describing the lack of increasedcardiac output during hemodilution with natural (unmodified) hemoglobinare Sunder-Plassmann et al., Eur. J. Int. Care Med. 1, 37-42 (1975) andMoss et al., Surg. Gyn. Ob., 142, 357-362 (1976).

Hemodilution with modified hemoglobin that has been polymerized also hasfailed to increase cardiac output. See Vlahakes et al., J. Thorac.Cardiovasc. Surg., 100, 379-388 (1990) which describes the hemodilutionof sheep with polymerized bovine hemoglobin prepared by BiopureCorporation; Rausch et al., supra (assigned to Biopure Corporation),which describes similar experiments; and Gould et al., Ann. Surg., 211,394-398 (1990) and Hobbhahn et al., Acta Anaesthesiol. Scand., 29,537-543 (1985) which describe hemodiluting baboons and dogs,respectively, with polymerized human hemoglobin solutions. Thus,hemodilution with both natural and modified hemoglobin has failed toincrease cardiac output.

Because cardiac output does not increase upon dilution of the blood withhemoglobin, body tissues are required, as one compensatory mechanism, toextract more oxygen from the diluted blood to prevent tissue damage fromhypoxia. However, such compensatory mechanisms have real limits in vivo.The heart, for instance, normally functions at about 95% maximal oxygenextraction levels and thus is only capable of increasing oxygenextraction about 5% (assuming 100% efficiency is possible). In the caseof hemodilution with albumin, even though arterial oxygen content isdecreased, oxygen delivery is essentially maintained at baseline levelsbecause of low viscosity blood that causes flow (cardiac output) toincrease proportionally. Therefore, hemodilution with hemoglobin offersno physiological or clinical advantage over hemodiluting with albumin.In fact, oxygen delivery is suboptimal during hemodilution withhemoglobin. This leads us to the central idea of the invention. Ifcardiac output or whole-body flow were allowed to increase as blood wasdiluted with hemoglobin then oxygen delivery would be clinicallysuperior to hemodilution with albumin because of the added arterialoxygen content provided by the plasma hemoglobin colloid.

The mechanism of unchanged cardiac output during hemodilution withhemoglobin may be caused by an inactivation of endogenous nitric oxide(NO), also called endothelium-derived relaxing factor (EDRF), which isan important regulator of blood vessel diameter. For nearly 100 hundredyears, it has been known that hemoglobin inactivates NO that has beendiffused to the blood via the lungs. Only recently has it beendiscovered that NO also forms biochemically in vivo. There is, however,uncertainty as to whether oxyHb directly reacts with NO or whether anindirect oxyHb-mediated product such as superoxide is responsible. Anoverview of the possible molecular interactions of oxyHb with NO andrelevant compounds is give below.

Initially, the reaction equations between NO and oxyHb/deoxyHb aresimple yielding

oxyHb+NO→metHb+NO₃ ⁻

deoxyHb+NO→NOHb

where metHb is ferric(Fe³⁺)hemoglobin, NO₃ ⁻ is nitrate and NOHb isnitrosylhemoglobin. However, NO₃ ⁻ can react with deoxyHb to yield:

2deoxyHb+NO₃ ⁻+H₂O→2metHb+NO₂ ⁻+2OH⁻

and nitrite (NO₂ ⁻) can react with oxyHb or deoxyHb to yield:

2oxyHb+NO₂ ⁻+H₂O→2metHb+NO₃ ⁻+2OH₂ ⁻

oxyHb+NO₂ ⁻+2H⁺→metHb+NO₂ +H₂O₂

deoxyHb+NO₂ ⁻+H₂O←→½[metHb+NO]+½[NOHb]+2OH⁻¹

where NO₂ is nitrogen dioxide and H₂O₂ is hydrogen peroxide. The freeenergy of activation (ΔG) of the last equation is rather low (−21.23kJ/mole) and therefore metHb . . . NO can be reduced to deoxyHb.However, in vivo experiments have shown that nitrite exposure results inabout an equal amount of NOHb and metHb formed in the blood.Furthermore, the ΔG of equation 2 is low (−46.02 Kj/mole) and in thepresence of O₂ will proceed according to the ΔG of equation 1 (≈−170Kj/mole). Finally, metHb and H₂O₂ can react:

metHb+H₂O₂→ferrylhemoglobin+H₂O

to produce a spectrophotometrically detectable red compound known asferryl(Fe⁺⁺⁺⁺)hemoglobin. In many of the above reactions, heme orchelatable iron can be substituted for Hb.

Under physiological conditions, i.e., in an oxygenated and heatedaqueous with a pH of about 7.4, NO is rapidly converted to nitrogendioxide:

2NO+O₂→2NO₂

Nitrogen dioxide is quite reactive, and in aqueous solutiondisproportionates to nitrate and nitrite as:

2NO₂→N₂O₄+H₂O→NO₃ ⁻+NO₂ ⁻+2H⁺

The decomposition of NO can also occur via:

NO₂+NO→N₂O₃+H₂O→2NO₂ ⁻+2H⁺

The abnormally rapid oxidation of oxyHb by NO is consistent with oxyHbserving as a superoxide (O₂ ⁻) donor, where

oxyHb+NO→metHb+ONOO⁻→NO₃ ⁻

or as recent studies propose, that NO is inactivated by other sources ofO₂ ⁻ yielding:

NO+O₂ ⁻→ONOO⁻+H⁺→HO.+NO₂→NO₃ ⁻+H⁺

where ONOO⁻ is peroxynitrite. Hydroxyl radical (HO.) can further reactwith NO₂ ⁻ to form NO₂. Peroxynitrate (O₂NOO⁻) and ONOO⁻ are alsosuspected intermediates in the autocatalytic oxidation of oxyHb by NO₂and NO, respectively.

Considering the autoxidation of oxyHb:

oxyHb←→metHb+O₂ ⁻

O₂ ⁻+oxyHb→metHb+O₂+H₂O₂

These last two equations are slow (the rate constants are 4-6×10³ M⁻¹sec⁻¹). A currently-popular hypothesis is that O₂ ⁻ is converted in thepresence of iron to the highly toxic HO. radical via the superoxidedriven Fenton reaction:

O₂ ⁻+Fe³⁺→Fe⁺⁺+O₂

2O₂ ⁻+2H⁺→H₂O₂+O₂

H₂O₂+Fe⁺⁺→HO.+OH⁻+Fe³⁺

Whether oxyHb+H₂O₂ forms HO. has not been proven. Furthermore, ferriciron (Fe³⁺) is sparingly soluble under physiological conditions and,therefore, must be chelated to heme, ferritin, etc to remain insolution. As with metHb, heme-Fe³⁺ may also react with H₂O₂ to form theferryl-heme radical (.Fe⁴⁺-heme).

Finally, rather than binding to heme, inactivation of endogenous NO mayoccur via its reaction with the thiol groups of oxyHb. Recently, it wasshown that NO circulates in mammalian plasma primarily as an S-nitrosoadduct on the thiol groups of serum albumin.

The biochemical pathway/effect of NO production and metabolism isbelieved to comprise the following, L-arginine→NO synthase→NO→guanylatecyclase→cyclic GMP→decreased blood vessel diameter. Conceivably,hemoglobin could decrease blood vessel diameter by inactivating anypoint of the pathway. Furthermore, hemoglobin does not effect thediameter of veins and arteries equally. Equal effect on these twovascular systems is necessary to increase blood flow or cardiac outputas observed during albumin hemodilution. Therefore, two mechanisms mustbe synchronized in order for cardiac output (and oxygenation) to bemaximized during hemodilution with hemoglobin: 1) molecular interventionof in vivo hemoglobin chemistry and 2) hemodynamic responsiveness invenous and arterial circulations.

There thus continues to exist a need in the art for new methods, andcompositions, useful for hemodilution with hemoglobin which increasecardiac output (oxygen delivery) and give this colloid a clinicaladvantage over non-oxygenated colloids such albumin.

SUMMARY OF THE INVENTION

The present invention provides therapeutic compositions for hemodilutionwith hemoglobin products that superaugment the oxygenating capacity ofthe circulatory system when using these products. The cardiacoutput-increasing compositions comprise hemoglobin and guanosine3′:5′-cyclic monophosphate (cyclic GMP) generating compounds. Methods oftreatment for diseases and medical, conditions requiring/indicating useof a superoxygenating compositions as hemodiluents, blood substitutes,plasma expanders, or resuscitative fluids are described. For use intherapeutic compositions of the present invention, the hemoglobinproducts may be modified to prevent rapid clearance from theintravascular space in vivo. For example, the hemoglobin products may becross-linked, chemically modified with compounds such as polyethyleneglycol or encapsulated (for example, in liposomes, glucose polymers orgelatin).

Therapeutic compositions according to the invention were formulated toreverse the vasoconstriction of hemoglobin products and to therebyfacilitate the flow-increasing effects of diluted blood (augmentedcardiac output). The compositions comprise either hemoglobin and cyclicGMP generating compounds or hemoglobin chemically coupled to cyclicGMP-generating compounds. Hemoglobin and excess nitric oxide werediscovered to exhibit a combined pharmacology resulting in increasedcardiac output in mammals. See Rooney et al., FASEB Journal, 5(4),Abstract 158 (1991).

Appropriate cyclic GMP-generating compounds contemplated by the presentinvention include, for example, nitric oxide precursors which areendogenous to mammals (e.g., L-arginine, lysine, glutamate, ornithine)and engineered compounds which contain nitric oxide groups (e.g., thenitrovasodilators sodium nitroprusside, organic nitrates,S-nitrosothiols, sydnonimines and furoxans) or which cause the releaseof nitric oxide in vivo (e.g., the endothelium-dependent vasodilatorsacetylcholine, bradykinin, adenine nucleotides and substance P) or anycompound which may directly or indirectly activate guanylate cyclase(dihydropyridines and related nitrovasodilator-dihydropyridine hybridstructures).

Cyclic GMP-generating compounds may be coupled to hemoglobin by chemicalprocesses which selectively attach the compounds to reactive amino(e.g., lysine residues), carboxyl (e.g., glutamate residues) orpreviously thiolated-amino groups on the hemoglobin surface. Nativethiol and disulfide groups of hemoglobin that play an importantstructural or functional role should be avoided.

Typically, a bifunctional reagent such as an imidoester may be used tocouple a cyclic GMP-generating compound to reactive hemoglobin groups.In some cases, a group on the cyclic GMP-generating compound may beactivated prior to incubation with hemoglobin. For example, a carboxylgroup on the cyclic GMP generating compound may be activated with acarbodi-imide for coupling to amino groups on the hemoglobin surface.Appropriate coupling reactions may involve other reagents, for example,sodium cyanoborohydride, carboxi-imides, succinic anhydride, thiols,N-hydroxysuccinimide and dithiothreitol.

The chemical coupling of the hemoglobin and the cyclic GMP-generatingcompound may involve a reversible or an irreversible bond. See, forexample, the coupling reactions in Carlsson et al., Biochem. J., 173,723-737 (1978) and Martin et al., J. Biol. Chem. 249, 286-288 (1981). Itmay be useful to react the β or α chain lysines or the amino terminalresidues of hemoglobin with agents that increase relative oxygendissociation [see Chatterjee et al., J. Biol. Chem., 261, 9929-9937(1986)] before coupling the hemoglobin to the cyclic GMP-generatingcompounds.

The therapeutic hemoglobin compositions of the present invention may bedescribed as “blood component substitutes.” In addition to hemoglobinand cyclic GMP-generating compounds, the compositions may comprisephysiologically acceptable plasma substitutes. Suitable plasmasubstitutes are linear polysaccharides (e.g., dextrans, gum arabicpectins, balanced fluid gelatin, and hydroxyethyl starch), polymericsubstitutes (e.g., polyethylene oxide, polyacrylamide, polyvinylpyrrolidone, polyvinyl alcohol, ethylene oxide-propylene glycolcondensate), aqueous solutions (e.g., Lactated Ringers and saline),coacervates (composed of fatty acids, phospholipids, glycerates orcholesterol, for example) and colloidal substitutes (e.g., albumin).

The therapeutic compositions according to the present invention areuseful for treatment of diseases or medical conditions in whichintravascular or intraosseous administration of a resuscitative fluid orblood plasma expander is indicated/required. Resuscitative fluids andblood plasma expanders are required for treatment of diseases andmedical conditions in which there is significant blood loss, hypotensionand/or a need to maximize the availability of oxygen to the bodytissues. Examples of such diseases and medical conditions arehemorrhagic hypotension, septic shock, cardio-pulmonary bypass, sicklecell and neoplastic anemias, plasma and extracellular fluid loss fromburns, stroke, angioplasty, cardioplegia, radiation therapy, acutemyocardial infarction, and both routine and lengthy surgical procedures.

Methods of treating such diseases and medical condition according to thepresent invention comprise the step of hemodiluting a mammal with apharmaceutically effective amount of a hemoglobin composition accordingto the present invention. Hemodilution with the compositions isperformed in a manner conventional in the art, for example, as describedin Messmer et al., Prog. Surg., 13, 208-245 (1974). The presentinvention also contemplates that cyclic GMP generating compounds maysimply be infused peri-hemodilution with a hemoglobin product. Themethods of treatment of the invention are particularly useful intreating humans.

The contents of this specification are submitted as sufficient objectivefactual evidence that the invention claimed is not prima facia obviousunder 35 U.S.C § 103 and that the invention claimed could not have beenexpected to be achieved by one of ordinary skill since the art was notof public record at the time the invention was made.

The prior art only demonstrated that hemodilution, i.e. substitution ofred cell mass, with hemoglobin failed to provide increased cardiacoutput or oxygen delivery. Exactly how this happens or the expectedmethods of reversing this result were not known in the art at the timethe invention was made. The prior art did not know that hemoglobin-basedmaterial selectively inhibits vasomotor activity in arteries that isdifferent from the inhibited vasomotor activity by hemoglobin-basedmaterial in veins. It was not known by anyone skilled in the art, exceptthe applicant, that coupling of hemoglobin and nitric oxide groups(nitrovasodilators) and particularly hemoglobin and NO from Nanitroprusside (SNP) would increase cardiac output. The prior art knewfor over 100 years that hemoglobin-based material binds nitric oxide,that hemoglobin-based material is a potential biocolloid, and that SNPis a vasodilator, however, the invention claimed was not prima faciaobvious and was pure luck. In fact, the applicant expected that the NOdonor used from SNP would not work since, in control animals orpatients, SNP dilates arteries and veins equally, decreases bloodpressure and rarely increases cardiac output unless heart rateincreases. Therefore, the unexpected result was that SNP can not dilateveins in the presence of hemoglobin-based material thereby not affectingpreload. This result discovered that hemoglobin does not effect arteriesand veins equally. Blood pressure was also little affected, anotherunexpected result. Because of these unexpected results, stroke volume(i.e. cardiac output) was allowed to increase inversely proportional tothe reduced viscosity component of afterload.

This invention will not work with every NO donor. For example,nitroglycerin and cyclic GMP analogues, in their present commercialformulation, will dilate both veins and arteries, with or withouthemoglobin-hemodilution. There is no increase in cardiac output becauseboth preload and afterload are reduced. This is further evidence thathemoglobin-based material affects arteries and veins by differentmechanisms and that one skilled in the art could not expect this result,or, one skilled in the art could not expect that the invention claimedcould be achieved.

The invention uses a series of related dihydropyridines to selectivelyprovide various time-controlled oxygen deliveries. These compounds areclassically slow channel calcium antagonists that preferentially dilatearteries to lower blood pressure over specific time periods. In theabsence of hemoglobin products, the dihydropyridine compounds generatecyclic GMP with increases of 20-70% from baseline levels. In thepresence of hemoglobin products, the dihydropyridine compounds providehemodynamics similar to those achieved with SNP, i.e. little effect onblood pressure and preload but appropriate reversal of hemoglobinantagonism in arteries. The fact that these compounds are unexpectedlysimilar to SNP in dilating arteries preferentially over veins suggeststhat hemoglobin-based material probably antagonizes the guanylatecyclase enzyme rather than scavenging NO directly or antagonizing the NOsynthase enzyme. Oxygen delivery, therefore, is similar to SNP but hasthe additional advantage of being time-controlled.

The invention uses cyclic GMP generating compounds as the principlemeans of increasing oxygen delivery with any hemoglobin-based material.The evidence from the SNP and dihydropyridine studies and other studiesof record presented in this communication clearly demonstrate thathemoglobin-based material inactivates or antagonizes more than oneNO→cyclic GMP mechanism, i.e. effects on NO→cGMP mechanisms in arteriesare not expected to be physiologically equivalent to those in veins.Furthermore, the reversal of hemoglobin inactivation or antagonism isdependent on more than one NO→cyclic GMP mechanism, i.e. reversal ofNO→cGMP antagonism in arteries is not expected to be physiologicallyequivalent to that in veins. Therefore, the invention selectively actson those NO→cGMP mechanisms to decrease afterload while having littleeffect on NO→cGMP mechanisms that control preload.

In summary, the specification is submitted as sufficient objectivefactual evidence, with unexpected results, that the invention claimed isnot prima facia obvious under 35 U.S.C § 103 and that the inventionclaimed could not have been expected to be achieved by one of ordinaryskill in the art since the art or knowledge of the selective vasoactivemechanisms of hemoglobin-based material and claimed compounds requiredfor control of these mechanisms was not known at the time the inventionwas made.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is bar graph representing the cardiac output (L/minute) ofcontrol, sodium nitroprusside-treated (SNP), albumin-hemodiluted (AB),hemoglobin-hemodiluted (Hb), and hemoglobin/sodiumnitroprusside-hemodiluted (HbSNP) mongrel dogs;

FIG. 1B is a bar graph representing the sum of regional blood flows(ml/minute) of control, sodium nitroprusside-treated (SNP),albumin-hemodiluted (AB), hemoglobin-hemodiluted (Hb), andhemoglobin/sodium nitroprusside-hemodiluted (HbSNP) mongrel dogs;

FIG. 1C is a bar graph representing the stroke volume (ml/beat)(computed from cardiac output) of control, sodium nitroprusside-treated(SNP), albumin-hemodiluted (AB), hemoglobin-hemodiluted (Hb), andhemoglobin/sodium nitroprusside-hemodiluted (HbSNP) mongrel dogs;

FIG. 1D is a bar graph representing the stroke volume (ml/beat)(computed from sum of regional blood flows) of control, sodiumnitroprusside-treated (SNP), albumin-hemodiluted (AB),hemoglobin-hemodiluted (Hb), and hemoglobin/sodiumnitroprusside-hemodiluted (HbSNP) mongrel dogs;

FIG. 2A is a bar graph representing the systolic aortic pressure (mm Hg)of control, sodium nitroprusside-treated (SNP), albumin-hemodiluted(AB), hemoglobin-hemodiluted (Hb), and hemoglobin/sodiumnitroprusside-hemodiluted (HbSNP) mongrel dogs;

FIG. 2B is a bar graph representing the diastolic aortic pressure (mmHg) of control, sodium nitroprusside-treated (SNP), albumin-hemodiluted(AB), hemoglobin-hemodiluted (Hb), and hemoglobin/sodiumnitroprusside-hemodiluted (HbSNP) mongrel dogs;

FIG. 2C is a bar graph representing the right atrial pressure (mm Hg) ofcontrol, sodium nitroprusside-treated (SNP), albumin-hemodiluted (AB),hemoglobin-hemodiluted (Hb), and hemoglobin/sodiumnitroprusside-hemodiluted (HbSNP) mongrel dogs;

FIG. 2D is a bar graph representing the left ventricular end-diastolicpressure (mm Hg) of control, sodium nitroprusside-treated (SNP),albumin-hemodiluted (AB), hemoglobin-hemodiluted (Hb), andhemoglobin/sodium nitroprusside-hemodiluted (HbSNP) mongrel dogs;

FIG. 3A is a bar graph representing the systemic vascular resistance(dyne·cm·s⁻⁵) of control, sodium nitroprusside-treated (SNP),albumin-hemodiluted (AB), hemoglobin-hemodiluted (Hb), andhemoglobin/sodium nitroprusside-hemodiluted (HbSNP) mongrel dogs;

FIG. 3B is a bar graph representing the systemic vascular hindrance(dyne·cm·s⁻⁵·cP⁻¹) of control, sodium nitroprusside-treated (SNP),albumin-hemodiluted (AB), hemoglobin-hemodiluted (Hb), andhemoglobin/sodium nitroprusside-hemodiluted (HbSNP) mongrel dogs;

FIG. 4A is a bar graph representing the regional blood flows (ml/minute)in the kidney (KI), gastrointestinal tract (GI), spleen (SP), pancreas(PA), liver/hepatic arteries (LI), lung/bronchial arteries (LU) andskeletal muscle (SM) of control (a), sodium nitroprusside-treated (b),albumin-hemodiluted (c), hemoglobin-hemodiluted (d), andhemoglobin/sodium nitroprusside-hemodiluted (e) mongrel dogs;

FIG. 4B is a bar graph representing the regional blood flows (ml/minute)in the skin (SK), bone (BO), right ventricle (RV), left ventricle (LV),septum (SE) and brain/cerebellum (BR) of control (a), sodiumnitroprusside-treated (b), albumin-hemodiluted (c),hemoglobin-hemodiluted (d), and hemoglobin/sodiumnitroprusside-hemodiluted (e) mongrel dogs;

FIG. 5A is a bar graph representing the fractional distribution ofcardiac output (%) in the kidney (KI), gastrointestinal tract (GI),spleen (SP), pancreas (PA), liver/hepatic arteries (LI), lung/bronchialarteries (LU) and skeletal muscle (SM) of control (a), sodiumnitroprusside-treated (b), albumin-hemodiluted (c),hemoglobin-hemodiluted (d), and hemoglobin/sodiumnitroprusside-hemodiluted (e) mongrel dogs; and

FIG. 5B is a bar graph representing the fraction distribution of cardiacoutput (%) in the skin (SK), bone (BO), right ventricle (RV), leftventricle (LV), septum (SE) and brain/cerebellum (BR) of control (a),sodium nitroprusside-treated (b), albumin-hemodiluted (c),hemoglobin-hemodiluted (d), and hemoglobin/sodiumnitroprusside-hemodiluted (e) mongrel dogs.

DETAILED DESCRIPTION

The following examples illustrate practice of the invention in treatingmammals with combinations of hemoglobin and cyclic GMP-generatingcompounds. The examples are not to be construed as limiting theinvention.

EXAMPLE I

As discussed previously, hemodilution with hemoglobin produces ahemodynamic profile of no increase in cardiac output, a profile which isuncharacteristic for hemodilution. Hemodilution with combinations ofhemoglobin and cyclic GMP-generating compounds, on the other hand,augments cardiac output as is illustrated below.

Twelve dogs were hemodiluted with a hemoglobin product according to thepresent invention either in the presence or absence of excessintravascular nitric oxide provided by sodium nitroprusside (SNP)infusion as the cyclic GMP generating compound. Hemodilution withalbumin of twelve dogs also either in the presence or absence of excessintravascular nitric oxide was performed as a control. Human albumin innormal saline was purchased as Albuminar-25 (25 mg %) from ArmourPharmaceutical Company (Kankakee, Ill.). The systemic and regionalhemodynamic responses of the twenty-four dogs were then measured. SNPwas chosen as a nitric oxide-associated compound because, unlike othernitrovasodilators, it is acts equally on both arteries and veins, itdoes not generate superoxide and it has no apparent requirement forcofactors or oxidative processes. See, Van Zwieten, Handbook ofHypertension: Pharmacology of Antihypertensive Drugs, 3, 308-330 (1984).The study was approved by the Loyola University Animal Care and UseCommittee and performed in accordance with the National ResearchCouncil's Guide for the Use of Laboratory Animals.

Hemoglobin Preparation

Canine hemoglobin (10 gm %) was purified as described in the parentapplication Ser. No. 07/849,610 filed on Mar. 11, 1992 and subsequentlypublished by Rooney et al., Anesthesiology, 79, 60-72 (1993). A 10 gm %solution of hemoglobin is optimal for use as a resuscitative fluid orblood plasma expander because the 10 gm % concentration corresponds tothe mass of red cells in blood (i.e., a hematocrit of 30 vol %) which isoptimal for whole body oxygen delivery (calculated from arterial bloodoxygen content×cardiac output). A 10 gm % solution of hemoglobin isoncotically active, has a lower viscosity than whole blood, and binds1.34 cc of oxygen per gram of hemoglobin at ambient oxygen pressures.

Various properties of the hemoglobin product are set out below in Table1.

TABLE 1 PROPERTY VALUE Total Hb concentration 10 gm % % methemoglobin <1% % carboxyhemoglobin < 0.5% % sulfhydrylhemoglobin Unknown %nitrosylhemoglobin Unknown Colloid osmotic pressure 40 mm Hg Viscosity ≈2.0 cp P₅₀ 24 mm Hg (pH 7.4) Bohr coefficient 0.54 (pH 7.2-7.4) Hillplot Normal Haldane effect Normal Davenport diagram Normal Oxygencontent 13.5 vol % Oxygen capacity 13.5 vol % pH 6.9 PCO₂ 15 mm Hg PO₂200 mm Hg Na 150 mmol/L K 3.4 mmol/L Ca⁺ 1.2 mmol/L Osmolality 310mOsmol/kg Dimers Unknown Monomer Unknown Free heme Unknown Free ironUnknown Trace metals Unknown

Results not presented in Table 1 are that methemoglobin levels werestable at ±0.4% for two weeks at 4° C., and that while hemoglobin dimersand monomers and free heme were not measured, in vitro studies haveshown that for hemoglobin concentrations of above about 1 to 2 gm %there is no detectable dissociation of hemoglobin tetramers [see Fanelliet al., Adv. Protein Chem., 19, 96-117 (1964)].

The hemoglobin product was also tested in vivo on dogs for commonadverse effects of therapeutic products which are administeredintravascularly. The hemodilution protocol used and the physiologictests performed are standard in the art and were similar to thatdescribed in Rabiner et al., J. Exp. Med., 86, 455-463 (1967);Sunder-Plassmann et al., supra; and Crystal et al., Anesth. Analg., 67,211-218 (1988) Briefly, the hemoglobin product was infused into a venousaccess of an anesthetized dog with simultaneous withdrawal of arterialblood on a one-to-one basis at about 25 ml/minute until hematocrit was50% of baseline (about 20 vol %). Tests for pyrogenic effects,hypotensive effects, arrhythmia, inotropic effects, bradycardia,tachycardia, hypovolemia, dysoria, and coagulopathy were all negative.

Surgical Preparation

Twenty-four conditioned, heartworm-free male mongrel dogs (20 to 30 kg)were anesthetized with sodium pentobarbital (30 mg/kg i.v.) followed byan i.v. maintenance dose of 4 mg/kg/hour. After intubation of thetrachea with a cuffed endotracheal tube, the dog was mechanicallyventilated (Siemens 900D Servoventilator) with 100% oxygen, tidalvolumes of 10 to 12 ml/kg and respiration at a rate to achievenormocarbia. These settings were not changed throughout the study.Sodium bicarbonate was not administered. The body temperature of the dogwas maintained at 39 C. with water-circulated heating pads.

The dog was placed supine and a polyethylene catheter (PE 200) wasinserted into the thoracic aorta via the left femoral artery formeasurement of blood pressure. Two small-bore (PE 90) heparin-filledcatheters of different lengths were placed in the abdominal aorta viathe right femoral artery to collect reference blood samples containingradioactive microspheres for measurement of regional blood flow.Wide-bore (PE 240) catheters were placed in the right femoral vein andin the right carotid artery, for isovolemic exchange transfusion and forthe administration of intravenous fluids and collection of arterialblood samples. A 5 french thermodilution catheter was advanced into thepulmonary artery via the right external jugular vein for measurement ofcardiac output and right atrial pressure. A Foley catheter was insertedinto the bladder for urine collection.

Under fluoroscopy, a 5 french volume-conductance catheter (MansfieldWebster) was inserted via the left carotid artery across the aorticvalve to the apex of the left ventricle to measure instantaneous volume.An 8/10 french Fogarty venous thrombectomy catheter was placed via theleft femoral vein into the inferior vena cava just above the diaphragmto produce occlusive-unloading of the left ventricle over severalcardiac cycles (20 seconds) during collection of pressure-volume data.

The animal was then placed on its right side and paralyzed withdoxacurium (0.05 mg/kg) to perform a left thoracotomy in the fourthintercostal space. The exposed lung was retracted with gauze. Five cmH₂O positive end-expiratory pressure was instituted to preventatelectasis. A small incision was made in the pericardium near the leftatrial appendage. The appendage was protracted and a PE 90 catheter, formicrosphere injection, inserted into the ventricle via pressureverification and then pulled back into the atrium. A 3 frenchmicromanometer-tipped pressure catheter (Millar) was then inserted viathe appendage into the left ventricle for pressure recording. Bothcatheters were secured with a ligature around the appendage and distallytaped to the animal. The exposed thoracic surface was covered withplastic film to prevent evaporation.

Measurements and Calculations

Continuous measurements of heart rate (HR), pulsatile aortic pressure,mean aortic pressure (MAP), left ventricular peak pressure (LVPP),rate-of-change of LVPP (dP/dt), LV end diastolic pressure (LVEDP), LVvolume, and right atrial pressure (RAP) were recorded on an analogthermal array recorder (Gould Model TA4000) and stored on a computerizeddata acquisition system (Halcom, Inc.) Cardiac output (CO) was measuredin triplicate using a Spectramed Hemoprol computer. Systemic vascularresistance (SVR) was calculated from (MAP−RAP)÷CO. Systemic vascularhindrance (SVH) was calculated from SVR÷η, where η is the apparentviscosity of blood in centipoise (cp). At high flow rates (shear rates≈200 s⁻¹) assumed in the aorta, η is 4.0 cp for hematocrit (Hct) of 40vol % and 2.1 cp for Hct of 20 vol %. Stroke volume was derived fromCO÷HR. LV stroke work (LVSW) was calculated from (systolicAoP−LVEDP)×SV×0.0136.

Blood pH, PCO₂, PO₂ and Na⁺, K⁺ and Ca⁺⁺ concentrations were measuredwith a Nova Stat Profile 1 analyzer (Waltham, Mass.). Plasma colloidosmotic pressure (COP) was determined before and after hemodilution witha Wescor 4400 Colloid Osmometer (Logan, Utah). The COP of 8% albumin was39.3±0.9 mm Hg. The COP of 10% hemoglobin was 40.8±1.0 mm Hg. Hematocritwas determined volumetrically. Hemoglobin (gm %), methemoglobin (%) andpercent oxygen saturation were measured with an InstrumentationLaboratories 482 CO-Oximeter (Lexington, Mass.). Hemoglobin oxygencontent was measured with the co-oximeter and added to the dissolvedoxygen (0.003×PO₂) to give total blood oxygen content (vol %).

Whole body oxygen extraction ratio (O₂ extr, %) was calculated fromarteria-mixed venous oxygen content difference C(a-v)O₂ divided by thearterial oxygen content (CaO₂). Whole body oxygen consumption (WBVO₂) inml/minute was determined using the Fick equation, WBVO₂=CO×C(a-V)O₂.Oxygen delivery (DO₂) in ml/minute was calculated from CaO₂×CO.

Catecholamines (pg/ml) were measured in arterial plasma usinghigh-performance liquid chromatography with electrochemical detection(RAS 400 Liquid Chromatograph (West Lafayette, Ind.). Arterial plasmalactate concentrations (meq/L) were measured enzymatically with an EasyST analyzer (E. Merck, Gibbstown, N.J.). Blood cyanide levels (mg/L)were measured spectrophotometrically by SmithKline Beecham ClinicalLaboratories (Schaumburg, Ill.).

Total blood volume was computed from plasma volume (indicator dilutionof iodinated I¹²⁵-albumin, Mallinckrodt Medical, Inc., St. Louis, Mo.)and whole body hematocrit.

Regional Blood Flows and Distribution of Cardiac Output Regional bloodflows (ml/minute/100 g tissue) were measured with the reference isotopetechnique using 15μ microspheres as described in detail in Crystal etal., supra. Briefly, prior to injection, microspheres labeled with Sc⁴⁶,Sr⁸⁵, Sn¹¹³ or Ce¹⁴¹were vortexed and sonicated. Approximately 30microcuries (1×10⁶ microspheres) were injected into the left atrium of adog. Beginning with each microsphere injection, duplicate referenceblood samples were collected at a constant rate (6 ml min⁻¹) for 3minutes from the femoral PE 90 catheters. Radioactivity of the duplicatesamples differed by less than 10%, indicating adequate mixing of themicrospheres in the left ventricular output. To maintain isovolemicconditions during reference sampling, a 5% albumin solution was infusedsimultaneously.

After the final injection of microspheres, the heart was stopped byintravenous injection of potassium chloride. Skin and bone (rib) weresampled from a shaved area distal to the thoracotomy. Skeletal musclesamples were taken from the hindlimb, back, forelimb and head. The GItract was excised from the esophageal sphincter to the anus. Allmesentery and omentum were trimmed. The stomach was separated from thetract. These and all other organs were weighed. Multiple samples weretaken from each organ and transferred to a tared counting tube.

The tissue and reference samples were weighed and analyzed forradioactivity with a gamma scintillation counter equipped with amultichannel analyzer (Packard Instrument, Downers Grove, Ill.). Isotopeseparation was accomplished by standard techniques of gammaspectroscopy. Values for organ blood flows (BF_(organ)) in ml min⁻¹ werecalculated from the equation BF_(organ)=ABF×(MC÷AC)×organ weight (g),where ABF is the rate of arterial reference sampling (ml/minute), MC isthe microsphere radioactivity (counts min⁻¹ g⁻¹) in the tissue samples,and AC is the total microsphere radioactivity (counts/minute) in thearterial reference samples. The fractional distribution of cardiacoutput to each organ was computed from BF_(organ)=ΣBF_(organ), whereΣBF_(organ) is the sum of all organ flows. Skeletal muscle, skin andbone weights were calculated as 40%, 9% and 8% of body weight,respectively.

Left Ventricular End-Systolic Elastance (E_(es))

Left ventricular contractility was determined from end-systolicelastance (Ees) using pressure-volume relationships according to themethods of Kass et al., Circulation, 79, 167-178 (1989). Briefly, acatheter with 11 electrodes spaced 1 cm from its distal end waspositioned in the ventricle so that its tip was at the apex (verifiedwith fluoroscopy). A weak electrical field (20 KHz, 0.03 mA RMS current)was generated through the LV cavity from the electrodes at the apex andat the aortic valve. Conductances measured between pairs of electrodeswithin the field provided a volume conductance measurement that includesthe actual ventricular volume plus an offset volume dependent onstructures surrounding the ventricular cavity (LV tissue, RV tissue andblood, and juxtapericardial tissue). The offset volumes were ignoredbecause only relative volume changes, not absolute volume measurements,were considered in the final analysis.

The volume signals were processed by a Leycom Sigma 5 signal conditioner(Stitching, Holland). An inferior vena caval occlusion varied preload tothe heart, during which the first 10-15 cardiac cycles (orpressure-volume loops) were collected. As preload decreased the area ofeach loop decreased. An algorithm was then used to find the end-systolicpressure-volume point of each loop. A linear regression line througheach point determined an equation, the end-systolic pressure-volumerelationship (ESPVR). The slope of the ESPVR, called the end-systolicelastance (E_(es)), is a load independent measure of global leftventricular contractility. Increases and decreases in E_(es) correspondto increases and decreased in contractility, respectively. Limitationsto this technique: the conductance offset volume could potentiallychange from cardiac cycle to cardiac cycle during an occlusion thusskewing the ESPVR in one direction or another. To test for this error wemade slow (20 second) and fast (5 second) occlusions during eitherend-inspiratory or end-expiratory pauses. We found no difference in themeasured E_(es) values determined in this manner.

Experimental Protocol

Upon arrival in the laboratory, typically all dogs had hematocrits of 45vol % or greater and filling pressures (LVEDP) of 5 mm Hg or less.Following cannulation all animals were hydrated with 5 mg % albumin toincrease filling pressure (LVEDP) above 5 mm Hg and to bring hematocritto near 40 vol %. Twelve dogs underwent isovolemic exchange of blood for10 gm % hemoglobin to hematocrit 50% of baseline. Twelve more dogs wereexchanged transfused with 8 gm % albumin in order to compare effects ofhemodilution with hemoglobin to those of hemodilution with an inertcolloid having a comparable molecular weight (both hemoglobin andalbumin have molecular weights of about 65,000). The colloid pressure of10 gm % hemoglobin and 8 gm % albumin (both about 40 Torr) is abouttwice that of dog plasma and after in vivo dilution would be expected tosustain plasma volumes at baseline values or greater. The smaller weightfraction of albumin needed to obtain a colloid pressure similar tohemoglobin is due to differences in surface charge, molecular shape,intermolecular association and hydration properties of the two colloids.

Hemodilution was produced by a simultaneous isovolemic exchange of bloodfor hemoglobin or albumin (rate of 20 ml/minute, about 45 ml/kg).Following the exchange all measurements and samples were obtained within30 minutes. SNP was then infused in five of the dogs hemodiluted withalbumin and in ten of the dogs hemodiluted with hemoglobin. Theend-point for SNP infusion was to obtain a decrease in mean aorticpressure (MAP) of at least about 10 mm Hg but not more than about 50 mmHg. The total dose of SNP varied (0.75-1.5 mg/kg) depending on the time(15-25 minutes) needed to reach the endpoint, record hemodynamic dataand to draw reference microsphere and other blood samples. Blood volumemeasurements were made after SNP infusion was discontinued.

Effects of Infusion of SNP Alone

A total of eight dogs, randomly selected from either the hemoglobin oralbumin group, were given SNP (2.3±0.4 μg kg⁻¹ minute⁻¹ i.v.) beforehemodilution with hemoglobin or albumin in order to establish thehemodynamic effects of SNP on these dogs. Baseline measurements weremade, then SNP was infused to decrease mean aortic pressure (MAP) by 40%to about 90 mm Hg. Once MAP stabilized (≈5-10 min), hemodynamicmeasurements, blood gases and other samples were obtained.

Cardiac output and stoke volume, computed via thermodilution or via thesum of regional blood flows, are shown in FIGS. 1A-1D. There was nosignificant difference in values obtained by the two computationmethods.

Moderate hypotensive doses of SNP (2.3±0.3 μg kg⁻¹ min⁻¹) had no effecton cardiac output or stroke volume (See FIGS. 3A-3B) but systolic,diastolic and mean aortic pressures were decreased about 34%. See Table2 below wherein “n” values represent the number of dogs treated with SNPalone (SNP), albumin alone (AbHD), hemoglobin alone (HbHD) or hemoglobinin combination with SNP (HbHD+SNP).

Left ventricular dP/dtmax decreased 44% as peak pressure decreased 33%,however LV elastance (contractility) was not changed from control values(See Table 2). Systemic vascular resistance and vascular hindrance bothdecreased approximately 31% (FIGS. 3A-3B), while right atrial pressureand left ventricular end-diastolic pressure decreased 55% and 73%respectively (FIGS. 2A-2D).

The foregoing results indicate that the decrease in blood pressure wasdue entirely to reductions in afterload and preload caused by decreasesin arteriolar and venous tone, respectively. Left ventricular strokework decreased 37%. Heart rate was not affected by these doses of SNP.

Blood parameters (arterial pH, PCO₂, PO₂, hemoglobin, blood volume,etc.) and whole body oxygenation were not significantly different fromcontrol values. See Table 2, and Tables 3 and 4 below wherein “n” valuesrepresent the number of dogs treated with SNP alone (SNP), albumin alone(AbHD), hemoglobin alone (HbHD) or hemoglobin in combination with SNP(HbHD+SNP).

Regional blood flows and the fractional distribution of cardiac outputto various organs were not affected by the moderate hypotensive doses ofSNP. (See FIGS. 4 and 5)

TABLE 2 Control SNP AbHD HbHD HbHD + SNP Variable n = 24 n = 8 n = 12 n= 12 n = 10 MAP (mm Hg) 134 ± 3 85 ± 3 122 ± 4 136 ± 6  110 ± 6 LVPP (mmHg) 153 ± 3 104 ± 8 150 ± 3 154 ± 6  139 ± 5 LV dP/dtmax 1669 ± 938 ±1753 ± 1713 ± 1887 ± 171 (mm Hg/sec) 88 118 137 76 LV Elastance 4.78 ±4.26 ± 5.26 ± 4.91 ±  4.24 ± 0.55 (mm Hg ml) 0.27 0.73 0.35 0.58 LVSW (gm/beat) 27.9 ± 17.7 ± 45.3 ± 29.2 ±  47.8 ± 4.71 1.5 1.5 3.5 3.4 heartrate 160 ± 4 161 ± 9 167 ± 6 153 ± 5  153 ± 7 (beats/min) Blood Volume1885 ± 1834 ± 1856 ± 1892 ± 1805 ± 118 (ml) 81 75 102 125

TABLE 3 Control SNP AbHD HbHD HbHD + SNP Parameter n = 24 n = 8 n = 12 n= 12 n = 10 pH 7.39 ± 7.33 ± 7.35 ± 7.38 ± 7.36 ± 0.02 0.01 0.02 0.010.01 PCO₂ (mm Hg) 35 ± 1 33 ± 1 37 ± 2 34 ± 2   35 ± 2 PO₂ (mm Hg) 390 ±301 ± 439 ± 411 ±  424 ± 33 21 36 33 31 Hematocrit 42 ± 1 40 ± 1 20 ± 121 ± 1   20 ± 1 (vol %) Na⁺ (mmol/L) 151 ± 1 152 ± 1 152 ± 1 152 ± 1 152 ± 1 K⁺ (mmol/L) 3.4 ± 3.5 ± 3.3 ± 3.7 ±  3.6 ± 0.1 0.1 0.2 0.1 0.2C⁺⁺ (mmol/L) 1.27 ± 1.22 ± 1.17 ± 1.15 ± 1.12 ± 0.08 0.02 0.03 0.03 0.05Total Hb 14.7 ± 14.0 ± 7.1 ± 11.5 ± 10.0 ± 0.1 (g/100 ml) 0.3 0.6 0.20.5 Total MetHb (%) 0.8 ± 0.5 ± 0.8 ± 0.8 ±  1.0 ± 0.09 0.04 0.09 0.060.07 Plasma Hb — — — 4.6 ±  3.4 ± 0.1 (g/100 ml) 0.2 Plasma MetHb — — —1.5 ±  1.5 ± 0.17 (%) 0.16 Plasma COP 18.7 ± 18.9 ± 22.5 ± 22.8 ± 20.9 ±0.6 (mm Hg) 0.6 0.6 0.7 0.8

TABLE 4 Control SNP AbHD HbHD HbHD + SNP Parameter n = 24 n = 8 n = 12 n= 12 n = 10 CaO₂ 19.7 ± 18.8 ± 10.0 ± 14.9 ± 13.2 ± 0.3 (ml/100 ml) 0.40.5 0.2 0.4 CvO₂ 14.2 ± 13.0 ± 6.8 ± 8.8 ±  9.7 ± 0.7 (ml/100 ml) 0.60.7 0.5 0.6 C(a-v)O₂ 5.1 ± 5.3 ± 2.7 ± 6.1 ±  3.3 ± 0.3 (ml/100 ml) 0.30.3 0.4 0.4 O₂ extr (%) 27 ± 3 30 ± 4 30 ± 2 45 ± 3   27 ± 4 DO₂(ml/min) 434 ± 404 ± 418 ± 302 ±  518 ± 38 18 35 25 22 WBVO₂ (ml/min)122 ± 8 119 ± 126 ± 125 ±  131 ± 14 15 10 11

In general, the systemic and regional effects of SNP infusion beforehemodilution were in agreement with other studies. See Hoka et al.,Anesthesiology, 66, 647-652 (1987); Wang et al., Anesthesiology, 46,40-48 (1977); Fan et al., Anesthesiology, 53, 113-120 (1980); Gelman etal., Anesthesiology, 49, 182-187 (1978); and Kien et al., Anesth.Analg., 66, 103-110 (1987).

In summary, intravenous infusion of 2.3±0.4 μg kg⁻¹ min⁻¹ of SNP beforehemodilution decreased MAP about 37% with no change in heart rate orcardiac output. There was no significant effect of SNP treatment onpre-hemodilution baseline data, i.e., dogs treated with SNP and dogs nottreated with SNP had similar hemodynamic profiles before hemodilution.

Effects of Hemodilution with Albumin

Hemodilution with albumin to a hematocrit 50% of control (20±1 vol %)caused cardiac output to increase to approximately 177% of control as isshown in FIGS. 1A-1D. Proportional increases occurred in stroke volumeas heart rate did not change. Left ventricular stroke work increasedmarkedly. (See Table 2) Hemodilution reduced systemic vascularresistance to about 54% of control but did not change systemic vascularhindrance (FIGS. 3A-3B). Mean aortic, right atrial and LV end-diastolicpressures were not changed. Thus the reduction in systemic vascularresistance was directly proportional to the apparent decrease inviscosity (assumed 50% of control) rather than to changes in arteriolartone. Stroke volume and cardiac output increased because the viscositycomponent of afterload was reduced allowing more complete emptying ofthe ventricle.

Hemodilution with albumin caused an approximate 50% reduction inhemoglobin concentration (14.7±0.3 to 7.0±0.2 g/100 ml) and arterialoxygen content (19.7±0.4 to 10.0±0.2 ml/100 ml) (See Tables 3 and 4).The arterial-mixed venous oxygen content difference decreased 47% whileoxygen extraction ratio was unchanged. Total body oxygen delivery andwhole body oxygen consumption were not changed from control values.Arterial blood gases, electrolytes and plasma catecholamines were withinthe control range (See Table 3 and Table 5 below). Hemodilution withhyperoncotic 8 gm % albumin caused plasma colloid osmotic pressure toincrease 20% yet there was no significant increase in blood volume. (SeeTables 2 and 3)

Compared to control and post-SNP infusion blood flows, regional bloodflows after hemodilution with albumin were significantly increased(about 80%) through various organ beds (see FIGS. 4A-4B). The increasedflows in the kidney, GI tract (stomach, small and large intestine,colon), liver (hepatic artery), lung (bronchial), skeletal muscle, skin,bone and brain were in approximate proportion to the increased cardiacoutput (see FIGS. 5A-5B). There was, however, a redistribution of flowduring hemodilution from the spleen, which received a smaller fractionof the cardiac output, to the heart which received a greater fraction ofthe cardiac output (see FIGS. 5A-5B).

Effects of Hemodilution with Hemoglobin

When hematocrit was reduced to 50% of control during hemodilution withhemoglobin, cardiac output and stroke volume did not change from controlvalues (see FIGS. 1A-1D).

Similar to hemodilution with albumin, systemic pressures were notchanged from control values (see FIGS.

TABLE 5 Parameter Control SNP AbHD HbHD HbHD + SNP Norepinephrine 114 ±7 — 106 ± 6 125 ±  102 ± 14 pg/ml, n = 8 12 Epinephrine 217 ± 18 — 249 ±19 219 ±  252 ± 22 pg/ml, n = 8 14 Lactate  1.4 ± 0.2 — — 2.2 ±  2.7 ±0.4 meq/L, n = 6 0.3 Cyanide — — — 0.13 ± 0.46 ± 0.04 mg/L, n = 6 0.03

2A-2D and Table 2). Despite a decrease in apparent viscosity similar toalbumin hemodilution (assumed 50% of control), systemic vascularresistance was not changed from control values, however, systemicvascular hindrance increased almost 100% (see FIGS. 3A-3B). Leftventricular elastance and other hemodynamic parameters were not changedfrom control values. Thus, stroke volume and cardiac output were notincreased because the decreased viscosity component of afterload wasoffset by the increased hindrance component (SVH).

Whole blood hemoglobin concentration decreased only 22% duringhemodilution with hemoglobin (from 14.7±0.3 to 11.5±0.5 g/100 ml).Plasma hemoglobin (4.6 g/100 ml) comprised approximately 40% the totalwhole blood hemoglobin concentration (see Table 2). Arterial oxygencontent decreased only 24% (from 19.7±0.4 to 14.9±0.4 ml/100 ml) (seeTable 4). Despite the additional oxygen supplied by plasma hemoglobin,hemodilution and the unchanged cardiac output resulted in a 30% decreasein oxygen delivery. However, a 60% increase in oxygen extraction ratiomaintained oxygen consumption at baseline levels (Table 4). Arterial pH,electrolytes and plasma catecholamines were unchanged from controllevels.

Total blood methemoglobin was not changed significantly from control,although the infused hemoglobin (plasma) had a greater percentage ofmethemoglobin (see Table 2). The plasma methemoglobin level was notsignificantly different from that of the purified hemoglobin product.Arterial blood gases and electrolytes remained within the control range.Again, similar to the albumin solution, the hyperoncotic hemoglobinsolution caused plasma colloid osmotic blood pressure to increaseapproximately 22% yet there was no significant change in blood volume(see Tables 2 and 3). Some plasma hemoglobin dissociation was evident bythe presence of hemoglobin in the urine (hemoglobinuria). The amountexcreted varied but was always less than 1% of the approximately 100grams hemoglobin infused.

Changes in regional blood flows were measured and the results arepresented below in Table 6. (See also FIGS. 4A-4B)

TABLE 6 ORGAN BLOOD FLOW CHANGE FROM CONTROL Renal No GastrointestinalNo Spleen Yes (−39%) Pancreas No Liver No Lung Yes (−53%) Skin No BoneNo Skeletal muscle No Myocardium Yes (+80%) Brain Yes (+40%) Total (=Cardiac output) No

Thus, as described previously, in contrast to hemodilution with albumin,hemodilution with hemoglobin product did not augment cardiac output. AsTable 6 shows, though, a fraction of cardiac output going to the spleenand lung was redistributed to the heart and brain. (See also FIGS.5A-5B)

Hemodilution with Hemoglobin in Combination with SNP

The SNP dose (≈2 μg/kg/min i.v.) tested before hemodilution wascompletely ineffective following hemodilution with hemoglobin. Two-wayANOVA revealed a significant interaction between hemoglobin hemodilutionand SNP. Larger SNP doses (54.2±4.6 μg kg⁻¹ min⁻¹ i.v.) were needed todecrease MAP only 18% (see Table 2). However, during this infusioncardiac output increased to approximately 180% of control values (seeFIGS. 1A-1D). Proportional increases occurred in stroke volume as heartrate did not change.

In contrast to SNP infusion before hemoglobin hemodilution, SNP infusionduring hemoglobin hemodilution had no effect on preload (LVEDP) or rightatrial pressure (see FIGS. 2A-2D). Systemic vascular resistance andhindrance decreased about 50% from hemoglobin hemodilution to valuessimilar to albumin hemodilution (FIGS. 3A-3B). Thus, although adecreased vascular component (MAP) of afterload was evident, theincreased stroke volume and cardiac output during hemoglobinhemodilution along with SNP were primarily due to an unmasking of thereduced viscosity component of afterload as seen with albuminhemodilution.

Compared to hemoglobin hemodilution alone, during hemoglobinhemodilution along with SNP, oxygen delivery increased and extractionratio decreased thereby returning to control values (see Table 4).Oxygen consumption was unchanged. Arterial hemoglobin, methemoglobin,pH, electrolytes, plasma catecholamines, and lactate levels were notsignificantly different from hemoglobin hemodilution values (see Tables3 and 5). Blood cyanide levels were elevated but did not reach toxicconcentrations (see Table 4).

Similar to albumin hemodilution, regional blood flows after hemoglobinhemodilution in combination with SNP infusion were significantlyincreased through various organ beds as is indicated below in Table 7.(See also FIGS. 4A-4B)

TABLE 7 ORGAN BLOOD FLOW CHANGE FROM CONTROL Renal No GastrointestinalYes (+80%) Spleen No Pancreas Yes (+80%) Liver Yes (+80%) Lung Yes(+80%) Skin Yes (+80%) Bone Yes (+80%) Skeletal muscle Yes (+80%)Myocardium  Yes (+200%) Brain Yes (+80%) Total (= Cardiac output) Yes(+80%)

As is shown in Table 7, cardiac output was augmented by hemodilutionwith hemoglobin in combination with SNP. In this study, for about everyten molecules of hemoglobin, about one molecule of nitric oxide in theform of SNP was required to increase cardiac output for the shortduration (30 minutes) studied.

The increased blood flows in the GI tract, pancreas, liver, lung, skin,bone and brain were in approximate proportion to the increased cardiacoutput and thus the fraction of cardiac output to these organs was notchanged from control values (see FIGS. 5A-5B). There was, however, aredistribution of flow from the kidney and spleen which received smallerfractions of the cardiac output, to the skeletal muscle and heart whichreceived a greater fraction of the cardiac output (See FIGS. 5A-5B).Left ventricular myocardial flow increased to almost 500 ml/minuteindicating near maximal vasodilation of the coronary arteries.

Because cyanide is released during SNP metabolism the toxicity of thiscompound was a concern. Whole blood levels of cyanide (0.46 mg/L) in thepresent study did not reach toxic levels despite doses of SNP (0.8 to1.4 mg/kg per 30 minutes) that were near those reported to be dangerousin Michenfelder, Anesthesiology, 62, 415-421 (1985). Vasey et al.,Anesthesiology, 62, 415-421 (1985) describes the infusion a similar SNPdose (50.0 μg/kg/min or 1.5 mg/kg/30 minutes) and detected approximately1.8 mg/L whole blood cyanide. Consistent with lower plasma cyanidelevels, our dogs did not exhibit the severe acidosis (arterial pH−7.18±0.02) or elevated plasma lactate caused by doses of SNP greaterthan 1.0 mg/kg.

EXAMPLE II

To gain more direct evidence that the molecular mechanism governing thehemodynamic pharmacology of hemoglobin-hemodilution involvesinactivation or antagonism of NO, we conducted a study on systemicguanosine 3′:5′-cyclic monophosphate (cyclic GMP) production. Cyclic GMPis the end-product of the L-arginine→NO synthase→NO→ guanylatecyclase→cyclic GMP pathway. In a series of dog studies using the methodsdescribed in Example 1, plasma from arterial blood samples was examinedfor cGMP concentrations before and after hemodilution with eitheralbumin (Ab-HD or oxyhemoglobin (oxyHb-HD). In Table 8, hemodilutioncaused an increase in plasma cGMP levels, however, the response wassignificantly attenuated in oxyHb-HD.

TABLE 8 Condition Plasma cGMP [μg/ml] n = 5 Control 9.04 ± 0.62 Ab-HD23.78 ± 1.14* n = 5 Control 9.76 ± 0.70 oxyHb-HD  17.22 ± 1.02*† valuesare mean ± sem. *, P < 0.05 from respective control. †, from Ab-HD.

The data suggests that as blood is diluted, a signal triggers therelease of nitric oxide with subsequent cGMP formation. It is not clearwhether the signal is a humoral substance in plasma or a physicalcomponent such as PO₂, PCO₂, viscosity, etc. However, the physiologicalsignificance of increased cGMP formation may be to facilitate increasesin blood flow via arteriolar dilation. Since oxyHb-HD and Ab-HD diluteblood to the same extent, the attenuated cGMP formation in oxyHb-HD mustbe due oxyHb mediated interference with nitric oxide synthase, NOdirectly or guanylate cyclase activity.

The effects of administering a nitric oxide donor/cyclic GMP generator(Na nitroprusside) on plasma cyclic GMP levels and arterial bloodpressure are shown in Table 9 for control (non-hemodiluted), Ab-HD andoxyHb-HD animals.

TABLE 9 Condition Plasma cGMP [μg/ml] MAP, mm Hg n = 5 Control 9.11 ±0.55 160 ± 9  ≈2 μg/kg/min SNP 13.06 ± 0.82*  121 ± 6*  ≈8 μg/kg/min SNP17.80 ± 0.94*  80 ± 5* n = 5 Ab-HD 23.78 ± 1.14  138 ± 8  ≈4 μg/kg/minSNP 24.28 ± 1.22   75 ± 3* n = 5 oxyHb-HD 17.22 ± 1.02  142 ± 6 ≈50μg/kg/min SNP 26.44 ± 1.76* 127 ± 9 values are mean ± sem. *, P < 0.05from control, Ab-HD or oxyHb-HD

In the control studies (non-hemodiluted), therapeutic doses of SNPproduced inverse but proportional changes in plasma cGMP and meanarterial pressure. Clearly, the hypotension was mediated by vasodilationinduced by the cGMP formed as a result of nitric oxide release from SNP.In Ab-HD, however, SNP caused a similar hypotension but no additionalincrease in plasma cGMP concentration. The obvious question is: why wasspillover of CGMP to the plasma maximized during Ab-hemodilution alone?In the case of oxyHb-HD, additional spillover of CGMP was evident whenlarge but non-therapeutic SNP doses were administered. Again, it is notclear where in the NO→cGMP pathway does oxyHb exert its inhibitoryeffect.

In a small pilot study, we investigated the possibility that oxyHb wouldcause selective differences in regional cGMP production (Table 10). Anin vivo method that is based on direct microdialysis of the organ tissuewas used to examine the regional interstitial production of cGMP. Themicrodialysis technique uses a small dialysis fiber (250 μm diameter)that is inserted into the tissue and perfused to sample extracellularmetabolites.

TABLE 10 Cyclic GMP levels (μg/ml) in regional interstitialmicrodialysate before hemodilution (CTRL), during hemodilution witheither albumin (AbHD) or hemoglobin (HbHD), and then during a subsequentinfusion of Na nitroprusside (2 and 50 μg/kg/min, respectively). (n =04) KI GI SP LI LU MU BO HE BR CTRL 9.42 6.56 12.58 7.74 10.48 11.32 9.147.80 13.28 ±0.96 1.10 1.78 0.98 1.96 1.60 1.00 0.92 1.36 Ab 20.54*18.12* 22.60* 14.24* 22.46* 22.24* 18.12* 21.56* 24.16* ±1.68 1.50 2.181.66 2.92 1.52 1.50 2.08 1.22 SNP 24.24* 27.78*† 21.60* 18.96* 23.64*24.81* 26.08*† 24.59* 26.76* ±4.74 1.95 2.90 3.48 4.96 1.49 2.18 3.404.32 (n = 4) KI GI SP LI LU MU BO HE BR CTRL 9.71 5.00 10.36 8.82 9.8810.70 9.00 7.54 13.62 ±0.72 0.50 0.94 1.04 1.12 1.18 0.94 0.66 0.48 Hb17.51* 12.68* 16.40* 20.34* 18.74* 17.98* 18.64* 14.44* 19.94* ±1.581.14 1.32 1.94 2.02 1.30 1.72 2.11 1.76 SNP 28.36*† 23.26*† 19.38*22.78* 26.38* 24.40*† 18.48* 20.20*† 27.65*† ±2.93 2.22 1.16 2.63 1.891.66 2.65 1.29 3.73 KI, kidney; GI, gastrointestinal tract; SP, spleen;LI, liver; LU, lung; BO, bone; HE, heart; BR, brain. Values are mean ±SE. *P < 0.05 vs. control; † P < 0.05 vs. respective HD.

With exception of GI tract and brain, there were no significantdifferences in cGMP levels between organ beds. With greater sample sizesit can be estimated that more regional difference will be evident. Ingeneral, the regional interstitial data were not remarkably differentfrom the plasma measurements.

We conducted a separate pilot study to test if administration of anitric oxide synthase inhibitor (L-nitro arginine methyl ester, i.e.L-NAME) during Ab-HD would cause hemodynamics and cGMP production tomimic oxyHb-HD. Approximately 6 mg/kg L-NAME was administered i.v. bolusduring Ab-HD. Significant increases in MAP and decreases in plasma cGMPwere evident (Table 11).

TABLE 11 cGMP, μg/ml MAP, mm Hg CO, ml/min Control 8.74 ± 0.70 134 ± 6 2201 ± 134  Ab-KD 22.04 ± 1.01* 126 ± 6  3710 ± 255* L-NAME  17.38 ±0.98*† 152 ± 8*†  2413 ± 1721† n = 3 in each group; values are mean ±sem. *, P < 0.05 from control; †, from Ab-HD; L-NAME = 6 mg/kg/min

Cardiac output was also returned to control (pre-hemodilution) levels.These results support our hypothesis that the unchanged cardiac outputof oxyHb-HD is caused by interference of the NO-cGMP system. However, itwas not clear where in the NO→cGMP pathway does oxyHb causeinactivation.

In another pilot study, we examined if infusion of a nitric oxidesubstrate (N-benzoyl-L-arginine ethyl ester, i.e. BAEE) would overridethe hypothesized NO scavenging by oxyHb and thereby mimic Ab-HDhemodynamics and CGMP production shown in Tables 8-11. While BAEEadministration increased plasma cGMP levels and caused significantvasodilation as evidenced by the decreased mean arterial pressure,cardiac output was not increased (Table 12).

TABLE 12 cGMP, μg/ml MAP, mm Hg CO, ml/min Control 8.56 ± 0.80 143 ± 7 2978 ± 134 oxyHb-HD 17.80 ± 0.95* 146 ± 5  2880 ± 245 BAEE  24.44 ±1.05*† 108 ± 8*† 2376 ± 233 n = 3; values are mean ± sem. *, P < 0.05from control. †, from oxyHb-HD; BAEE = 200-400 μg/kg/min

Comparing these results to those obtained with SNP administration (seeExample 1 and Tables 8-11), it becomes apparent that oxyHb selectivelyis more potent at opposing NO-CGMP activation by SNP in venous relativeto arteriolar beds. In other words, SNP was able to reduce thearteriolar afterload produced by oxyHb-mediated arteriolar constrictionbut did not affect oxyhb-mediated venoconstriction or preloading. Hence,cardiac output increased. This is atypical of SNP, which classicallydilates both venous and arteriolar beds with little effect on cardiacoutput. Whereas in the case of BAEE administration, oxyHb causedrelatively little opposition to arterial or venous dilation by BAEE.This was evidenced by the decreased MAP and unchanged cardiac output(Table 12). It may that the high membrane permeability of BAEE wasresponsible for its unimpeded participation in cGMP formation (Table12). Different

More unexpected results were obtained when BAEE was used to reverse theeffects of L-NAME in AB-HD dogs. With BAEE, the original hemodynamics ofAb-HD alone were expected to be evident, i.e., increased cardiac output.This was not the case, cardiac output was not reversed to the highlevels of Ab-HD alone. These data and the hemoglobin-BAEE evidencesuggest that hemoglobin may bind or antagonize to a greater extent theguanylate cyclase receptor in arteries compared to veins. The exactmechanism is not obvious. Nor, is the method of reversing hemoglobinmediated effects on cardiac output obvious to one skilled in the art.

EXAMPLE III

Hemodilution with oxyhemoglobin colloid (oxyHb) produces avasoconstriction that attenuates cardiac output (CO) and oxygen delivery({dot over (D)}O₂). The vasoconstriction is caused by an interference ofguanylate cyclase activity and is selectively greater in veins comparedto arteries. Vasodilators that potentiate guanylate cyclase activitypreferentially in arteries compared to veins sustain preload but reduceafterload and, hence, increase CO and maximize {dot over (D)}O₂ ofoxyHb-hemodilution. Dihydropyridine compounds are classically calciumchannel antagonists but are now discovered to “from oxyHb-HD;BAEE=200-400 μg/kg/min” these compounds have varying terminal half-lifeeliminations (t½ in hours) they may provide a time-controlledsuperaugmentation of CO and {dot over (D)}O₂ during hemodilution withhemoglobin products.

Anesthetized dogs were isovolemically hemodiluted to ≈20% Hct with 10%oxyHb combined physically, or infused separately, with dihydropyridinecompounds (DHP) of varying t½ (hours): DHP(≈8-14 h), DHP(≈14-24 h) andDHP(≈24-45 h). The end point was to minimize the amount of each compoundneeded to reduce oxyHb arteriolar constriction (evidenced by a ↓ in meanBP) while increasing cardiac output ≈50% or more (comparable toalbumin-hemodilution). Systemic hemodynamics and guanylate cyclaseactivity (cyclic GMP formation) in mixed venous blood are reportedduring baseline and following hemodilution.

TABLE 13 Effect of hemodilution with oxyHb-dihydropyridine compositionson systemic hemodynamics and blood cyclic GMP formation. oxyHbDHPoxyHbDHP oxyHbDHP Baseline (8-14 h) (14-24 h) (24-45 h) [DHP] ≈ 100 μg ≈100 μg ≈ 5000 μg Duration 0.5-1 hour 1-2 hours 2-4 hours MAP, mmHg 143 ±6  118 ± 3*  115 ± 4*  140 ± 8  HR, b/min 135 ± 9  114 ± 4*  112 ± 4* 128 ± 10* CO, ml/min 2510 ± 194 4353 ± 182* 4066 ± 144* 4741 ± 228* SV,ml 18.9 ± 1.6 38.9 ± 2.4* 36.6 ± 1.8* 37.4 ± 2.0* cGMP, ng/min 268 ± 34758 ± 58* 937 ± 89* 587 ± 26* n = 6 in each DHP group; values are mean ±sem; *, P < 0.05 vs. respective baseline.

All three classes of oxyHb-DHP compositions increased CO durationssomewhat proportional to their elimination half-life. However, therelatively short duration of effects for these long-acting DHP compoundssuggests that oxyHb may compete for a site near the calcium channelreceptor. The significant increase in mixed venous cGMP also supports apartial mechanism of DHP pharmacology that involves activation of thecGMP pathway, a result especially important to reversing oxyHbvasoconstriction. These studies suggest that the oxyHb-DHB compositionsprovide a range of time-controlled superaugmentation of CO, andconsequently ⁰DO₂, which could be applied to specific patients, i.e.,shorter duration compositions for trauma-related blood replacement andlonger durations for blood conservation in surgical procedures.

What is claimed is:
 1. A method for controlling intravascular blood flowand blood oxygen delivery to tissues of a body of a human or animalsubject, comprising: intravenously administering an effective amount ofan oxygen containing hemoglobin blood substitute to said subject; andadministering to said subject an effective amount of a cyclic guanosinemonophosphate-generating compound for a time and under conditionseffective to alter blood flow and oxygen to said tissues a hemoglobinblood substitute to said subject; and administering to said subject acyclic guanosine monophosphate-generating compound.
 2. The method ofclaim 1, wherein said administration to said subject effects arterialdilation to a greater extent than venous dilation.
 3. The method ofclaim 1, wherein said administration to said subject effects increasedstroke volume.
 4. The method of claim 1, wherein cardiac output ofoxygenated blood in said subject is increased.
 5. The method of claim 1,wherein said administration effects reversal of hemoglobin-derivedantagonism of cyclic GMP generation.
 6. The method of claim 1, whereinsaid blood flow and oxygen delivery have rates that are time-controlled.7. The method of claim 1 wherein said administration of said hemoglobinand cyclic GMP-generating compound provides reduced vasoconstriction insaid subject.
 8. The method of claim 1, wherein blood flow in one ormore regions of the body increases following said administration ofcyclic GMP-generating compound and said hemoglobin substitute.
 9. Themethod of claim 8, wherein said one or more regions of the body areselected from the group consisting of the gastrointestinal organs,pancreas, liver, lung, skin, bone, skeletal muscle, myocardium andbrain.
 10. The method of claim 1 wherein said cyclic GMP-generatingcompound is chemically coupled to said hemoglobin.
 11. The method ofclaim 10, wherein said chemical coupling comprises binding of saidcyclic GMP-generating compound to lysine residues, glutamate residues orthiolated amino groups on hemoglobin.
 12. The method of claim 1 whereinsaid cyclic GMP-generating compound is selected from the groupconsisting of dihydropyridines, nitric oxide donors, nitric oxideprecursors, nitric oxide releasers, and cyclic GMP analogs.
 13. Themethod of claim 12 wherein said cyclic GMP-generating compound isselected from the group consisting of sodium nitroprusside, organicnitrates, S-nitrosothiols, syndonimines, furoxanes, andnitrovasodilator-dihydropyridine hybrids.
 14. The method of claim 12wherein said cyclic GMP-generating compound is selected from the groupconsisting of L-arginine, N-benzoyl-L-arginine ethyl ester,N-substituted arginine analogs, lysine, glutamate and ornithine.
 15. Themethod of claim 12 wherein said cyclic GMP-generating compound isselected from the group consisting of acetylcholine, bradykinin, adeninenucleotides, dihydropyridines and nitrovasodilator-dihydropyridinehybrids.
 16. A method for controlling intravascular blood flow and bloodoxygen delivery to tissues of a body of a human or animal subject,comprising: intravenously administering an effective amount of an oxygencontaining hemoglobin blood substitute to said subject; andadministering to said subject an effective amount of a nitricoxide-associated compound for a time and under conditions effective toalter blood flow and oxygen to said tissues.
 17. The method of claim 16,wherein said administration to said subject effects arterial dilation toa greater extent than venous dilation.
 18. The method of claim 16,wherein said administration to said subject effects increased strokevolume.
 19. The method of claim 16, wherein cardiac output of oxygenatedblood in said subject is increased.
 20. The method of claim 16, whereinsaid administration effects reversal of hemoglobin-derived antagonism ofnitric oxide.
 21. The method of claim 16, wherein said blood flow andoxygen delivery have rates that are time-controlled.
 22. The method ofclaim 16 wherein said administration of said hemoglobin and nitricoxide-associated compound reverses vasoconstriction in said subject. 23.The method of claim 16, wherein blood flow in one or more regions of thebody increases following said administration of nitric oxide-associatedcompound and said hemoglobin substitute.
 24. The method of claim 23,wherein said one or more regions of the body are selected from the groupconsisting of the gastrointestinal organs, pancreas, liver, lung, skin,bone, skeletal muscle, myocardium and brain.
 25. The method of claim 16wherein said nitric oxide-associated compound is chemically coupled tosaid hemoglobin.
 26. The method of claim 25 wherein said chemicalcoupling comprises binding of said nitric oxide-associated compound tolysine residues, glutamate residues or thiolated amino groups onhemoglobin.
 27. The method of claim 16 wherein said nitricoxide-associated compound is a nitric oxide precursor, nitric oxidedonor, or nitric oxide releaser.
 28. The method of claim 27 wherein saidnitric oxide-associated compound is selected from the group consistingof L-arginine, N-benzoyl-L-arginine ethyl ester, N-substituted arginineanalogs, lysine, glutamate, and ornithine.
 29. The method of claim 27wherein said nitric oxide-associated compound is selected from the groupconsisting of sodium nitroprusside, organic nitrates, S-nitrosothiols,syndonimines, fuoxans, and nitrovasodilator-dihydropyridine hybridstructures.
 30. The method of claim 27 wherein said nitricoxide-associated compound is selected from the group consisting ofacetylcholine, bradykinin, adenine nucleotides, dihydropyridines andnitrovasodilator-dihydropyridine hybrids.